Bacteriophages: Update on Application As Models for Viruses in Water
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Bacteriophages: Update on application as models for viruses in water WOK Grabow Department of Medical Virology, University of Pretoria, PO Box 2034, Pretoria 0001, South Africa Abstract Phages are valuable models or surrogates for enteric viruses because they share many fundamental properties and features. Among these are structure, composition, morphology, size and site of replication. Even though they use different host cells, coliphages and Bacteroides fragilis phages predominantly replicate in the gastro-intestinal tract of humans and warm-blooded animals where enteric viruses also replicate. A major advantage of phages is that, compared to viruses, they are detectable by simple, inexpensive and rapid techniques. In view of these features, phages are particularly useful as models to assess the behaviour and survival of enteric viruses in the environment, and as surrogates to assess the resistance of human viruses to water treatment and disinfection processes. Since there is no direct correlation between numbers of phages and viruses, phages cannot to a meaningful extent be used to indicate numbers of viruses in polluted water. The presence of phages typically associated with human and animal excreta indicates the potential presence of enteric viruses. However, the absence of these phages from water environments is generally a meaningful indication of the absence of enteric viruses. This is because phages such as somatic coliphages, F-RNA coliphages and B. fragilis phages generally outnumber enteric viruses in water environments, and they are at least as resistant to unfavourable conditions including those in water treatment and disinfection processes. However, using highly sensitive molecular techniques viruses have been detected in drinking water supplies which yielded negative results in conventional tests for phages. Initially, data on phages were rather confusing because a wide variety of techniques was used. However, techniques for the detection of phages are being standardised internationally. This applies in particular to somatic and F-RNA coliphages, and B. fragilis phages, which are most commonly used in water quality assessment. Reliable and practical techniques now available include direct quantitative plaque assays on samples of water up to 100 ml, and qualitative tests on 500 ml or more using highly sensitive enrichment procedures. Introduction morphological subunits called capsomeres. The capsomeres consist of a number of protein subunits or molecules called protomers. Bacteriophages (phages) are viruses which infect bacteria. They Some phages also contain lipid and additional structures such as were discovered independently by Frederick W Twort in England tails and spikes. These features imply that in terms of composition, in 1915 and by Felix d’Herelle at the Pasteur Institute in Paris in structure and morphology, phages share many fundamental 1917 (Pelczar et al., 1988). Phages were the last of the three major properties with human viruses. For instance, F-RNA coliphages classes of viruses to be discovered during World War I. The other (Family Leviviridae) and enteroviruses such as polio viruses (Family two classes were the plant viruses and animal viruses. It was then Picornaviridae) both have an icosahedral capsid with a diameter of hoped that their ability to kill bacteria could be used for the about 25 nm and a single strand (ss)-RNA genome. Under the prevention and treatment of bacterial disease, but this did not prove electron microscope F-RNA coliphages and enteroviruses are successful due to the rapid selection of resistant bacteria (Goyal et hardly distinguishable (Fig. 1). In addition, F-RNA coliphages and al., 1987). However, phages eventually turned out to have major enteroviruses are both excreted by humans. For these reasons other benefits, notably as models or surrogates for human viruses coliphages are valuable models or surrogates for human enteric in basic genetic research as well as water quality assessment. viruses. As a result of these similarities, the behaviour of F-RNA Phages proved to be most valuable tools in research on viruses coliphages as well as other phages, resembles that of enteric viruses because compared with the human, animal, plant and even insect much closer than faecal bacteria such as coliforms commonly used hosts of other viruses, phages are easily and rapidly cultivated in as indicators of faecal pollution. The same applies to properties laboratories which are not particularly demanding with regard to such as removal by water treatment processes and resistance to space, facilities, and equipment (Pelczar et al., 1988). Research on disinfection processes. However, there are differences which limit the basic genetic properties of phages led to the development of an the indicator value of phages. For instance, electrostatic charges on entirely new science - that of molecular biology - which allowed phages may differ from those on enteric viruses, which affect unprecedented advancements in all the biological and medical important properties such as adsorption to solid surfaces. This has sciences. In addition, the way all viruses reproduce was first implications for features like behaviour in the environment, and the indicated by work with phages (Ackermann, 1969). efficiency of recovery by techniques based on adsorption-elution principles. Structure and morphology of phages Phage replication Phages basically consist of a nucleic acid molecule (genome) surrounded by a protein coat (capsid). The capsid is made up of Phages and enteric viruses can replicate only inside host cells, which in the case of phages are susceptible bacteria, and in the case of enteric viruses are susceptible mammalian cells. Phages use the ((012) 319-2351; fax (012) 325-5550; e-mail: [email protected] Received 29 February 2000. ribosomes, protein-synthesising factors, amino acids, and energy- Available on website http://www.wrc.org.za ISSN 0378-4738 = Water SA Vol. 27 No. 2 April 2001 251 Inoviridae and Leviviridae. These features have major implications for the utilisation of phages as models/surrogates for human viruses. For instance, under certain optimal conditions host bacteria, notably heterotrophic bacteria, may support the replication of somatic phages in water environments. However, in environmental waters, even in sewage, conditions rarely if ever meet requirements for the production of fertility fimbriae. An important limiting factor is the temperature of at least 30°C. This implies that for all practical purposes replication of male-specific phages in environmental waters is most unlikely, while somatic coliphages may under circumstances multiply in certain water environments (Grabow et al., 1980). Optimal conditions for the replication of both somatic and male-specific phages typically prevail in the gastro-intestinal tract of humans and other warm-blooded animals. Since human enteric viruses are released into the environment almost exclusively from the gastro-intestinal tract of humans, phages which infect typical enteric bacteria such as E. coli, resemble human viruses with regard to origin and release into the environment. This strongly supports the value of phages, notably coliphages and Bacteroides fragilis phages, as models/surrogates for enteric viruses. Among phages, the F-RNA male-specific phages are theoretically probably the best models/surrogates for enteric viruses because: like enteric viruses they almost exclusively originate from the faeces of humans and other warm-blooded animals; like enteric viruses they fail to multiply in the environment; and in terms of composition, structure and size they closely resemble human enteric viruses. However, coliphages are excreted at all times by a certain percentage of all humans and other warm-blooded animals (Grabow Figure 1 et al., 1995), whereas enteric viruses of human health concern are Electron micrograph of F-RNA coliphages (Family Leviviridae). Size of about 25 nm and morphology are similar to that of human excreted almost exclusively by humans during infection which enteroviruses like polio virus. Note attachment of the phages to may last for a few days to a few weeks. This implies that the bacterial fertility fimbriae which have receptor sites for these incidence of human enteric viruses in the environment is subject to phages. The thicker rods are bacterial flagellae without variables such as the epidemiology of viral infections, outbreaks of receptor sites. infections in communities, vaccination against viruses, seasonal changes, and other variables which do not affect the excretion of generating systems of the host cell to replicate, and hence, phages coliphages. In addition, human enteric viruses and coliphages are can multiply only in metabolising host bacteria (Goyal et al., 1987). not excreted in the same numbers and not for the same periods of Some phage species have fewer than 10 genes and use almost all of time. Consequently there is rarely, if ever, a direct correlation the cellular functions, whereas others have 30 to 100 genes and are between the numbers of any coliphages and any enteric viruses in less dependent on the host. A few of the large phage particles have water environments at any time. so many of their own genes that, for certain functions such as DNA Phages are divided into two groups according to their mode of replication, they need no host genes (Freifelder, 1987). Phages can replication. Virulent (lytic) phages typically proceed with replication only infect certain bacteria. The